PROJECT SUMMARY: The applicant?s laboratory discovered that the assembly of the ESX-1 translocon in the cytoplasmic membrane regulates gene expression. The long-term goal of the applicant is to understand the biological significance of ESX-1-dependent changes in gene expression. The applicant proposes that the assembly of the ESX-1 trans- locon elicits gene expression pathways that couple phagosomal lysis and adaptation to cytoplasmic exposure. The overall objective of this proposal is to explore how the ESX-1 translocon regulates gene expression, which will contribute to addressing the applicant?s long-term goal. The central hypothesis is that there is an uncharac- terized transcription response network connecting the ESX-1 translocon to widespread changes in gene expres- sion, potentially linking phagosomal lysis and cytoplasmic adaptation. The rationale is that the successful com- pletion of the proposed work will provide insight into the fundamental mechanisms linking lysis of the phagosomal membrane by ESX-1 with changes in gene expression. The central hypothesis will be tested by following these specific aims: 1) Identify the transcription factor network that regulates ESX-1-dependent gene expression. 2) Define the genes required for ESX-1-dependent signal transduction. Under the first aim, the applicant proposes to use genetic, molecular and biochemical approaches to identify transcription factors and genes in the tran- scriptional network that responds to the ESX-1 translocon. Under the second aim, the applicant proposes to use genetic and molecular approaches to identify and characterize genes and signaling pathways that connect the translocon in the cytoplasmic membrane to changes in gene expression. The expected outcome following the successful completion of the proposed aims will be to define factors and pathways required for ESX-1-dependent changes in mycobacterial gene expression. This contribution will be significant because it will provide fundamen- tal insight into the regulation of bacterial factors that promote interaction with the host during the initial phases of infection, positively impacting our ability to prevent diagnose and treat TB. The proposed research is technically and conceptually innovative. Previous work on ESX-1 has focused on the mechanisms of secretion by and the effects of the ESX-1 system on the host. The proposed work focuses on the role of the ESX-1 system in regu- lating transcriptional response networks, which has not been previously considered. To the applicant?s knowledge, considering the assembly of the ESX-1 translocon as a signal that elicits changes in gene expression is a new idea. Finally, the combined multi-pronged approach to identify the mechanisms connecting the ESX-1 translocon to gene expression is innovative. Defining the molecular mechanisms underlying the control of gene expression by the ESX- translocon will advance the field by providing new mechanisms regulating mycobacterial gene expression.